Title
A continuous assay for a-methylacyl-coenzyme A racemase using circular dichroism
Author
Dahmane Ouazia, Stephen L. Bearne
Year
2010
Journal
Analytical Biochemistry
Abstract
α-Methylacyl-coenzyme A racemase (AMACR) catalyzes the epimerization of (2R)- and (2S)-methyl branched fatty acyl-coenzyme A (CoA) thioesters. AMACR is a biomarker for prostate cancer and a putative target for the development of therapeutic agents directed against the disease. To facilitate development of AMACR inhibitors, a continuous circular dichroism (CD)-based assay has been developed. The open reading frame encoding AMACR from Mycobacterium tuberculosis (MCR) was subcloned into a pET15b vector, and the enzyme was overexpressed and purified using metal ion affinity chromatography. The rates of MCR-catalyzed epimerization of either (2R)- or (2S)-ibuprofenoyl-CoA were determined by following the change in ellipticity at 279 nm in the presence of octyl-β-d-glucopyranoside (0.2%). MCR exhibited slightly higher affinity for (2R)-ibuprofenoyl-CoA (Km = 48 ± 5 μM, kcat = 291 ± 30 s−1), but turned over (2S)-ibuprofenoyl-CoA (Km = 86 ± 6 μM, kcat = 450 ± 14 s−1) slightly faster. MCR expressed as a fusion protein bearing an N-terminal His6-tag had a catalytic efficiency (kcat/Km) that was reduced 22% and 47% in the 2S → 2R and 2R → 2S directions, respectively, relative to untagged enzyme. The continuous CD-based assay offers an economical and efficient alternative method to the labor-intensive, fixed-time assays currently used to measure AMACR activity.
Instrument
J-810
Keywords
Circular dichroism, Kinetics, Stereochemistry, Biochemistry